Novel mutations in BRCA2 intron 11 and overexpression of COX-2 and BIRC3 mediate cellular resistance to PARP inhibitors

Several poly(ADP ribose) polymerase (PARP) inhibitors (PARPi) have been approved for Cancer therapy; however, intrinsic and acquired resistance has limited their efficacy in the clinic. In fact, Cancer cells have developed multiple mechanisms to overcome PARPi cytotoxicity in even a single Cancer cell. In this study, we generated three PARPi-resistant BRCA2-deficient pancreatic Capan-1 variant cells using olaparib (Capan-1/OP), talazoparib (Capan-1/TP), and simmiparib (Capan-1/SP). We identified novel mutations in intron 11 of BRCA2, which resulted in the expression of truncated BRCA2 splice isoforms. Functional studies revealed that only a fraction (32-49%) of PARPi sensitivity could be rescued by depletion of BRCA2 isoforms. In addition, the Apoptosis signals (phosphatidylserine eversion, Caspase 3/7/8/9 activation, and mitochondrial membrane potential loss) were almost completely abrogated in all PARPi-resistant variants. Consistently, overexpression of the anti-apoptotic proteins cyclooxygenase 2 (COX-2) and baculoviral IAP repeat-containing 3 (BIRC3) occurred in these variants. Depletion of COX-2 or BIRC3 significantly reduced apoptotic resistance in the PARPi-resistant sublines and reversed PARPi resistance by up to 70-72%. Furthermore, exogenous addition of prostaglandin E2, a major metabolic product of COX-2, inhibited PARPi-induced apoptotic signals; however, when combined with the BIRC3 inhibitor LCL161, there was significantly enhanced sensitivity of the resistant variants to PARPi. Finally, PARPi treatment or PARP1 depletion led to a marked increase in the mRNA and protein levels of COX-2 and BIRC3, indicating that PARP1 is a negative transcriptional regulator of these proteins. Together, our findings demonstrated that during the chronic treatment of cells with a PARPi, both BRCA2 intron 11 mutations and COX-2/BIRC3-mediated apoptotic resistance led to PARPi resistance in pancreatic Capan-1 cells.